JP7322545B2 - Gas concentration adjusting agent for culturing anaerobic bacteria and method for culturing anaerobic bacteria using the same - Google Patents

Description

The present invention relates to a gas concentration adjusting agent for culturing anaerobic bacteria and a method for culturing anaerobic bacteria using the same.

Cultivation of biological specimens such as tissues and cells performed in research fields or industrial fields of organisms, reproduction or biotechnology requires a gas environment different from the air atmosphere. For example, as a condition for maintaining the pH of the bicarbonate-buffered culture solution at pH 7.4, which is the same as the normal state of blood, it is necessary to set the atmospheric carbon dioxide concentration to about 5%. In addition, in many research fields, cell culture is performed under a low-concentration oxygen atmosphere similar to that in vivo.

Carbon dioxide gas incubators and the like are known as means for creating a gas environment with a high-concentration carbon dioxide atmosphere and a low-concentration oxygen atmosphere. Therefore, in recent years, a method using a gas concentration adjusting agent that utilizes an oxidation reaction of ascorbic acids has been widely used (see Patent Documents 1 and 2).

Japanese Patent No. 3818324 Japanese Patent No. 5682831

In culturing anaerobic bacteria, it is required to create a gas environment with a high-concentration carbon dioxide atmosphere. Therefore, it is desired to more efficiently increase the amount of carbon dioxide generated in the oxidation reaction of ascorbic acids.
The problem to be solved by the present invention is to provide a gas concentration adjusting agent for anaerobic bacteria culture that generates a large amount of carbon dioxide.

As a result of intensive studies, the present inventors have found that the amount of carbon dioxide generated increases when dehydroascorbic acid is blended, as compared to when ascorbic acids are blended. The present invention has been completed based on such findings.

That is, the present invention relates to the following.
<1> At least selected from the group consisting of (a) dehydroascorbic acid, (c) transition metal catalyst, (d) activated carbon, (e) alkali metal carbonate, alkali metal hydroxide and alkaline earth metal hydroxide 1, and (f) a gas concentration adjusting agent for anaerobic bacteria culture containing water.
<2> (b) The gas concentration adjusting agent for anaerobic bacteria culture according to <1> above, further comprising ascorbic acids.
<3> The gas concentration for anaerobic bacteria culture according to <2> above, wherein the molar ratio [(b)/(a)] of (b) ascorbic acids and (a) dehydroascorbic acid is 1.5 or less. regulator.
<4> A method for culturing anaerobic bacteria, comprising culturing anaerobic bacteria in the presence of the gas concentration adjusting agent according to any one of <1> to <3> above.
<5> A gas concentration adjusting agent package in which the gas concentration adjusting agent according to any one of the above <1> to <3> is packaged in a small bag with an air-permeable packaging material.

The gas concentration adjusting agent for anaerobic bacteria culture of the present invention generates a large amount of carbon dioxide, and can efficiently create a gas environment with a high-concentration carbon dioxide atmosphere and a low-concentration oxygen atmosphere.

An embodiment of the present invention will be described below. The content of the present invention is not limited to the embodiments described below.
In this specification, the term "A to B" regarding numerical values means "A or more and B or less" (when A<B) or "A or less than B" (when A>B). . Moreover, in the present invention, a combination of preferred aspects is a more preferred aspect.

[Gas concentration adjuster]
The gas concentration adjusting agent for culturing anaerobic bacteria of the present invention comprises (a) dehydroascorbic acid, (c) transition metal catalyst, (d) activated carbon, (e) alkali metal carbonate, alkali metal hydroxide and alkaline earth. It contains at least one selected from the group consisting of metal hydroxides and (f) water. The gas concentration adjusting agent may further contain (b) ascorbic acids.
Further, the gas concentration adjusting agent of the present invention includes (a) dehydroascorbic acid, (c) transition metal catalyst, (d) activated carbon, (e) alkali metal carbonate, alkali metal hydroxide and alkaline earth metal hydroxide. It is preferably used as a package in which a composition containing at least one selected from the group consisting of substances and (f) water is packaged with an air-permeable packaging material. The composition may further comprise (b) ascorbic acids.
The gas concentration adjusting agent of the present invention contains components (a) to (f) in a total amount of preferably 50% by mass or more, more preferably 60% by mass or more, and still more preferably 70% by mass or more.

(a) Dehydroascorbic acid The gas concentration adjusting agent of the present invention contains dehydroascorbic acid, which has both oxygen absorption ability and carbon dioxide generation ability, as a main ingredient of the oxygen absorption reaction.
Dehydroascorbic acid is a compound obtained by oxidizing ascorbic acid, and produces diketogulonic acid by hydrolysis. By adjusting an appropriate catalyst and reaction environment, decarboxylation and oxidation reactions are repeated to generate carbon dioxide and absorb oxygen. Dehydroascorbic acid can also be produced by oxidizing ascorbic acids.

In the gas concentration adjusting agent of the present invention, dehydroascorbic acid is preferably impregnated in activated carbon together with water from the viewpoint of oxygen absorption performance. Specifically, it is preferable to impregnate activated carbon as a porous carrier with an aqueous solution of dehydroascorbic acid in which dehydroascorbic acid is dissolved in water.

The gas concentration adjusting agent utilizes the oxidation reaction of dehydroascorbic acid to absorb oxygen in the atmosphere and adjusts its concentration, and uses the generated carbon dioxide to adjust the concentration of carbon dioxide in the atmosphere. Note that, in the oxidation reaction, carbon dioxide is theoretically produced in an equimolar amount or more to the consumed oxygen molecules. Due to the above principle, when the oxygen concentration is reduced, the carbon dioxide concentration is also increased accordingly.

(b) Ascorbic Acids The gas concentration adjusting agent of the present invention may contain ascorbic acids. Ascorbic acid is cheaper and more readily available than dehydroascorbic acid, and has both oxygen absorption ability and carbon dioxide generation ability as a main ingredient in the oxygen absorption reaction.
Ascorbic acids refer to L-ascorbic acid, its stereoisomers, salts and hydrates thereof. Examples of L-ascorbate include sodium L-ascorbate, potassium L-ascorbate, calcium L-ascorbate and the like. Stereoisomers of L-ascorbic acid include erythorbic acid (D-isoascorbic acid) and the like. Erythorbate includes sodium erythorbate, potassium erythorbate, calcium erythorbate and the like. One type of ascorbic acid may be used alone, or two or more types may be used in combination.

In the gas concentration adjusting agent of the present invention, ascorbic acids are preferably impregnated in activated carbon together with water from the viewpoint of oxygen absorption performance. Specifically, it is preferable to impregnate activated carbon as a porous carrier with an aqueous solution of ascorbic acids in which ascorbic acids are dissolved in water. In this case, the higher the concentration of the ascorbic acid aqueous solution, the less the amount of the porous carrier used. Therefore, it is preferable to select a compound having high water solubility as the ascorbic acid. Ascorbic acid or sodium ascorbate is preferable as the ascorbic acid from the viewpoint of solubility in water and availability. When ascorbic acid or sodium ascorbate is used, the concentration of the aqueous solution is preferably 40-55% by mass.

When the gas concentration adjusting agent of the present invention contains (b) ascorbic acids, the molar ratio [(b)/(a)] of (b) ascorbic acids and (a) dehydroascorbic acid is preferably 1.5 or less. , more preferably 1.2 or less, still more preferably 1.0 or less, still more preferably 0.7 or less, still more preferably 0.5 or less, still more preferably 0.1 or less. When culturing anaerobic bacteria, the composition is put into a gas-barrier sealed container and used, and the above composition is sufficient when the container is put.

(c) Transition Metal Catalyst The gas concentration regulator of the present invention contains a transition metal catalyst that accelerates the oxidation reaction of dehydroascorbic acid and ascorbic acids.
A transition metal catalyst is a catalyst having a metal compound such as a salt or oxide of a transition metal. Preferred transition metals are iron, manganese, zinc, copper and cobalt. Transition metal salts include transition metal halides and mineral salts, such as transition metal chlorides and sulfates. Representative examples include ferrous chloride, ferric chloride, ferrous sulfate, ferric sulfate, manganese chloride, zinc sulfate, copper sulfate, copper chloride, and anhydrous or hydrous salts of cobalt sulfate. Ferrous sulfate heptahydrate is preferred because it has good solubility in water and good blendability.

The content of the transition metal catalyst in the gas concentration adjusting agent is 1 to 30 parts by mass with respect to 100 parts by mass of dehydroascorbic acid and ascorbic acids in total, from the viewpoint of promoting the progress of the oxidation reaction of dehydroascorbic acid and ascorbic acids. is preferred, 5 to 25 parts by weight is more preferred, and 10 to 20 parts by weight is even more preferred.

(d) Activated carbon The gas concentration adjusting agent of the present invention contains activated carbon. Activated carbon functions as a carrier for impregnating the aqueous solution of dehydroascorbic acid and the aqueous solution of ascorbic acids, and also has a large specific surface area and a large contact area with air, thereby promoting the progress of the oxidation reaction.

As the activated carbon, for example, those manufactured by using sawdust, coal, coconut shells, etc. as raw materials, steam activation, chemical activation using zinc chloride, etc., can be used. Granular activated carbon is preferable because the activated carbon is used by loading an aqueous solution of dehydroascorbic acid, an aqueous solution of ascorbic acids, etc. on the activated carbon and filling it in a granular form in a sachet. The particle size of the granular activated carbon is preferably 0.1 to 2 mm, more preferably 0.5 to 1 mm, from the viewpoints of oxygen absorption performance and packing properties (fluidity).

The content of activated carbon in the gas concentration adjusting agent is preferably 50 to 400 parts by mass with respect to a total of 100 parts by mass of dehydroascorbic acid and ascorbic acids, from the viewpoint of oxygen absorption performance and packing performance, 75 to 300 parts by mass is more preferable.

(e) Alkali Metal Carbonate, Alkali Metal Hydroxide and Alkaline Earth Metal Hydroxide The gas concentration adjusting agent of the present invention comprises an alkali metal carbonate, an alkali metal hydroxide and an alkaline earth metal hydroxide. At least one selected from the group is included. At least one selected from the group consisting of alkali metal carbonates, alkali metal hydroxides and alkaline earth metal hydroxides accelerates the oxidation reaction of dehydroascorbic acid and ascorbic acids, and shifts the reaction field to an alkaline region. Used for control purposes.

As the alkali metal carbonate, water-soluble alkali metal carbonates such as sodium carbonate, sodium hydrogencarbonate and sodium carbonate hydrate are suitably used, with sodium carbonate being particularly preferred.
Examples of alkali metal hydroxides include potassium hydroxide and sodium hydroxide, with sodium hydroxide being preferred.
Alkaline earth metal hydroxides include calcium hydroxide and magnesium hydroxide.

Among alkali metal carbonates, alkali metal hydroxides and alkaline earth metal hydroxides, from the viewpoint of solubility in water when converted to a salt with ascorbic acid, the gas concentration adjuster of the present invention is an alkali metal It preferably contains a carbonate and/or an alkali metal hydroxide.
From the viewpoint of neutralizing hydrolysis products, the content of the alkali metal hydroxide in the gas concentration adjusting agent is preferably equimolar to the total molar amount of dehydroascorbic acid and ascorbic acids.
From the viewpoint of adjusting the carbon dioxide concentration, the content of the alkali metal carbonate in the gas concentration adjusting agent is preferably 10 to 200 parts by mass, and 50 to 200 parts by mass with respect to 100 parts by mass of dehydroascorbic acid and ascorbic acids in total. parts is more preferred, and 100 to 150 parts by mass is even more preferred.

(f) Water The gas concentration adjusting agent of the present invention contains water necessary for the oxidation reaction of dehydroascorbic acid and ascorbic acids to proceed.
It is preferable to impregnate activated carbon with water from the viewpoint of obtaining the gas concentration adjusting agent as a fluid solid matter. In the gas concentration adjusting agent of the present invention, water is preferably impregnated in activated carbon together with dehydroascorbic acid and ascorbic acids from the viewpoint of oxygen absorption performance. Specifically, it is preferable to impregnate activated carbon, which is a porous carrier, with an aqueous solution of dehydroascorbic acid or an aqueous solution of ascorbic acids in which dehydroascorbic acid or an ascorbic acid is dissolved in water. In addition, soluble components other than dehydroascorbic acid and ascorbic acids may be dissolved in water, or insoluble components may be dispersed.

The content of water in the gas concentration adjusting agent is preferably 30 to 500 parts by mass with respect to a total of 100 parts by mass of dehydroascorbic acid and ascorbic acids, from the viewpoint of promoting the oxidation reaction of dehydroascorbic acid and ascorbic acids. 50 to 300 parts by mass is more preferable, and 80 to 200 parts by mass is even more preferable.

(g) Other Components The gas concentration adjusting agent of the present invention may optionally contain components other than the components (a) to (f) described above within a range that does not impair the effects of the present invention.

(g1) Thermoplastic resin The gas concentration adjusting agent of the present invention may contain a thermoplastic resin in order to suppress excessive heat generation accompanying the progress of the oxygen absorption reaction (oxidation reaction of dehydroascorbic acid and ascorbic acids). The type of thermoplastic resin is not particularly limited, but for example, polyethylene, polypropylene, ethylene-vinyl acetate copolymer, elastomer or a mixture thereof can be used, particularly low molecular weight polyethylene having a molecular weight of 10000 or less, polypropylene or a mixture thereof. It is preferably used from the viewpoint that the softening point can be easily adjusted and the influence of odor is small.

The thermoplastic resin preferably has a particle size of 1 to 500 μm, more preferably 10 to 300 μm, from the viewpoint of miscibility with other components. Also, the softening point of the thermoplastic resin is preferably 90 to 125° C. from the viewpoint of suppressing heat generation more effectively.

From the viewpoint of promoting the oxidation reaction of dehydroascorbic acid and ascorbic acids, the content of the thermoplastic resin in the gas concentration adjusting agent is 100 to 1000 parts by mass with respect to a total of 100 parts by mass of dehydroascorbic acid and ascorbic acids. Preferably, 300 to 500 parts by mass is more preferable.

(g2) Aldehyde removing agent The gas concentration adjusting agent of the present invention may contain an aldehyde removing agent in order to remove aldehyde that is by-produced mainly with the progress of the oxidation reaction of dehydroascorbic acid and ascorbic acids. Various compounds such as amines are known as compounds having an aldehyde-removing ability. Ethylene urea, urea, Arginine, lysine hydrochloride, or polyallylamine is preferably blended, and ethylene urea, which is more effective in a smaller amount, is more preferable.

Aldehyde as used herein means a compound having one or more formyl groups in its molecule, that is, aldehydes. In the present invention, it typically means an aldehyde generated as a by-product in the process of absorbing oxygen or culturing bacteria, and the aldehyde is classified as an aldehyde in the chemical field as long as it has an adverse effect on culturing bacteria. Any thing is included if it is a thing. Specifically, for example, formaldehyde, acetaldehyde and the like are included.

From the viewpoint of efficiently and economically removing aldehyde, the content of the aldehyde removing agent in the gas concentration adjusting agent is 0.5 to 25 parts by mass with respect to 100 parts by mass of dehydroascorbic acid and ascorbic acids. It is preferably 1 to 10 parts by mass, and even more preferably 1 to 5 parts by mass.

(g3) Coating material The gas concentration adjusting agent of the present invention may have a coating material on the outside of the granules of the composition containing the components (a) to (f) described above. Examples of the coating material include porous particles such as activated carbon, zeolite, and silicate, from the viewpoint of adsorbing a small amount of odorous components generated along with the oxygen absorption reaction. Talc, magnesium stearate, calcium stearate and the like can be used from the viewpoint of improving the flowability of the gas concentration adjusting agent and facilitating the filling and packaging of the gas concentration adjusting agent in a packaging material. These auxiliaries can be used singly, or two or more of them can be used in combination, if necessary. In addition, these auxiliaries are readily available commercially.

(Method for producing gas concentration adjusting agent)
The method for producing the gas concentration adjusting agent of the present invention is not particularly limited. A method of mixing with an alkali metal carbonate and impregnating activated carbon can be mentioned. Alternatively, an aqueous ascorbic acid solution is prepared by dissolving ascorbic acids, a transition metal catalyst, an alkali metal hydroxide, etc., and an aqueous dehydroascorbic acid solution is prepared by dissolving dehydroascorbic acid, a transition metal catalyst, an alkali metal hydroxide, etc. Alternatively, a method of mixing these solutions with activated carbon and an alkali metal carbonate and impregnating the activated carbon with the solution is also available.

[Gas concentration adjusting agent package]
The gas concentration adjusting agent can also be made into a gas concentration adjusting agent package by packaging a composition containing each of the components described above in a packaging material that uses an air-permeable packaging material in whole or in part.

(packaging material)
As the packaging material, two sheets of air-permeable packaging material are pasted together to form a bag, and one sheet of air-permeable packaging material and one sheet of non-breathable packaging material are pasted together to form a bag-like form, For example, a sheet of air-permeable packaging material is folded, and edges other than the folded portion are sealed to form a bag.

Here, when the air-permeable packaging material and the non-air-permeable packaging material have a square shape, the packaging material is a bag-shaped material obtained by stacking two air-permeable packaging materials and heat-sealing the four sides of the packaging material. , A sheet of breathable packaging material and a sheet of non-breathable packaging material are superimposed and heat-sealed on four sides to form a bag, one sheet of breathable packaging material is folded, except for the folded part A case in which the sides are heat-sealed to form a bag is exemplified. Alternatively, the packaging material may be a bag-like material obtained by forming a tubular air-permeable packaging material into a tubular shape and heat-sealing both ends and a trunk portion of the tubular body.

(Breathable packaging material)
As a breathable packaging material, a packaging material that is permeable to oxygen and carbon dioxide is selected. Among them, those having an air resistance of 600 seconds or less, more preferably 90 seconds or less, as measured by the Gurley tester method, are preferably used. Here, the air resistance means a value measured by the method of JIS P8117 (1998). More specifically, it refers to the time required for 100 mL of air to permeate the air-permeable packaging material using a Gurley densometer manufactured by Toyo Seiki Seisakusho.

As the air-permeable packaging material, paper, non-woven fabric, and plastic film with air permeability are used. Examples of plastic films include films such as polyethylene terephthalate, polyamide, polypropylene, and polycarbonate, and films such as polyethylene, ionomer, polybutadiene, ethylene/acrylic acid copolymer, ethylene/methacrylic acid copolymer, and ethylene/vinyl acetate copolymer as sealing layers. can be used. These laminates can also be used as breathable packaging materials.

As a method for imparting air permeability, various methods such as perforation using cold needles and hot needles can be employed. When air permeability is imparted by perforation, the air permeability can be freely adjusted by the diameter, number, material, etc. of the holes to be perforated.

Also, the thickness of the laminated film is preferably 50 to 300 μm, particularly preferably 60 to 250 μm. In this case, compared with the case where the thickness is outside the above range, the packaging material can be made to have strength, heat-sealing property, and packaging suitability.

In order to maintain the function of the gas concentration adjusting agent package for a long period of time, it is preferable to store it in a gas-barrier container or bag before use and remove it from the gas-barrier container or bag before use. Further, when the gas concentration adjusting agent package is used for bacterial culture, it is preferable to sterilize the package in advance using γ-rays or the like.

[Method for culturing anaerobic bacteria]
The method for culturing anaerobic bacteria of the present invention is a method for culturing anaerobic bacteria in the presence of a gas concentration adjusting agent. Specifically, a culture container containing anaerobic bacteria and a culture medium and a gas concentration adjusting agent (preferably a gas concentration adjusting agent package) are placed in a gas barrier closed container, then sealed, and the closed container is used for bacterial culture. It can be carried out by standing under a temperature suitable for.

The medium used in the culture method of the present invention is not particularly limited, and commonly used media can be applied as they are. The concentration of aldehyde dissolved in the medium is preferably 2 mg/L or less, more preferably 1.5 mg/L or less, and still more preferably 1.0 mg/L or less, as conditions for bacterial culture.
Also, the culture temperature is preferably 20 to 45°C, particularly preferably 25 to 40°C.

The culture vessel is not particularly limited as long as air permeability with the outside of the vessel is ensured, and any volume, shape, material, etc. suitable for culture can be adopted. A culture vessel having a lid is preferably used, but even in this case, it is necessary to ensure air permeability with the outside of the vessel.

The method for culturing bacteria of the present invention is applied to culturing anaerobic bacteria. In the culture method of the present invention, the atmosphere in the gas-barrier sealed container before culture is not particularly limited and may be air, but from the viewpoint of culturing anaerobic bacteria, a nitrogen atmosphere is preferred.

The gas-barrier sealed container used in the bacterial culture method prevents the flow of gas inside and outside the container, and maintains for a long period of time the concentrations of oxygen and carbon dioxide formed by the introduced gas concentration adjusting agent. Containers made of glass, metal, plastic such as polycarbonate, etc. are often used, but gas barrier films and laminates thereof can also be used.

At this time, for the purpose of measuring the amount of aldehyde generated in the gas-barrier closed container and adjusting the humidity in the container, an open container containing distilled water was placed in the closed container. good too. Examples of the open-type container include a culture container, a beaker, a flask, and the like, and the same type of container as the culture container containing the bacteria and medium is preferable.

From the viewpoint of culturing anaerobic bacteria, it is preferable that the oxygen concentration in the closed container becomes 0.1% by volume or less within 24 hours. Also, the carbon dioxide concentration in the sealed container is preferably 11.5% by volume or more, more preferably 12% by volume or more, still more preferably 13% by volume or more, and even more preferably 15% by volume or more.

In bacterial culture, it is important to reach the desired oxygen concentration and carbon dioxide concentration in a short period of time. From the viewpoint of culturing anaerobic bacteria, the oxygen concentration at the beginning of the oxidation reaction of dehydroascorbic acid and ascorbic acids (3 hours after the start of the reaction) is preferably 0.50% by volume or less, and 0.30% by volume or less. More preferably, it is 0.15% by volume or less. The carbon dioxide concentration at the initial stage of the oxidation reaction of dehydroascorbic acid and ascorbic acids (3 hours after the start of the reaction) is preferably at least 11.5% by volume, more preferably at least 12% by volume, and even more preferably at least 13% by volume. , more preferably 15% by volume or more.

According to the culture method of the present invention, bacteria can be microscopically observed and transported under a suitable gas atmosphere without using a gas cylinder and a gas controller.

The present embodiment will be described in detail below using examples and comparative examples, but the present embodiment can be modified as appropriate as long as the effects of the present invention are exhibited. "Parts" in Examples and Comparative Examples means parts by mass unless otherwise specified.

Production example 1
(Preparation of ascorbic acid aqueous solution)
Under a nitrogen atmosphere, 30.6 g of L-ascorbic acid, 46.2 g of 15% by mass sodium hydroxide aqueous solution, and 4.8 g of ferrous sulfate heptahydrate are mixed to prepare a 2.13 mmol/g ascorbic acid aqueous solution. bottom.

Production example 2
(Preparation of dehydroascorbic acid aqueous solution)
Under a nitrogen atmosphere, 4.5 g of dehydroascorbic acid, 6.9 g of 15% by mass sodium hydroxide aqueous solution, and 0.7 g of ferrous sulfate heptahydrate are mixed to prepare a 2.13 mmol/g dehydroascorbic acid aqueous solution. bottom.

Example 1
In a nitrogen atmosphere, 10 g of activated carbon, 15 g of polyethylene powder, and 4.7 g of sodium carbonate were measured and mixed. To this powder, 10.1 g of an aqueous ascorbic acid solution and 10.1 g of an aqueous dehydroascorbic acid solution were added and mixed to produce an oxygen absorber composition. An aluminum bag was filled with 6.9 g of this oxygen absorber composition and sealed.
An aluminum bag containing the oxygen scavenger was enclosed in a nylon bag together with 750 mL of air, and the aluminum bag was opened in the nylon bag to start a deoxidation experiment. When the oxygen concentration and carbon dioxide concentration after 3 hours were measured with a gas chromatograph, the oxygen concentration was 0.20% by volume and the carbon dioxide concentration was 13.6% by volume. Further, when the oxygen concentration and carbon dioxide concentration after 24 hours were measured with a gas chromatograph, the oxygen concentration was 0.00% by volume and the carbon dioxide concentration was 13.7% by volume.

Example 2
In a nitrogen atmosphere, 10 g of activated carbon, 15 g of polyethylene powder, and 4.7 g of sodium carbonate were measured and mixed. To this powder, 20.3 g of dehydroascorbic acid aqueous solution was added and mixed to produce an oxygen absorber composition. An aluminum bag was filled with 6.9 g of this oxygen absorber composition and sealed.
An aluminum bag containing the oxygen scavenger was enclosed in a nylon bag together with 750 mL of air, and the aluminum bag was opened in the nylon bag to start a deoxidation experiment. When the oxygen concentration and carbon dioxide concentration after 3 hours were measured with a gas chromatograph, the oxygen concentration was 0.10% by volume and the carbon dioxide concentration was 18.5% by volume. Further, when the oxygen concentration and carbon dioxide concentration after 24 hours were measured with a gas chromatograph, the oxygen concentration was 0.00% by volume and the carbon dioxide concentration was 18.6% by volume.

Comparative example 1
In a nitrogen atmosphere, 10 g of activated carbon, 15 g of polyethylene powder, and 4.7 g of sodium carbonate were measured and mixed. To this powder, 20.3 g of an aqueous ascorbic acid solution was added and mixed to produce an oxygen absorber composition. An aluminum bag was filled with 6.9 g of this oxygen absorber composition and sealed.
An aluminum bag containing the oxygen scavenger was enclosed in a nylon bag together with 750 mL of air, and the aluminum bag was opened in the nylon bag to start a deoxidation experiment. When the oxygen concentration and the carbon dioxide concentration after 3 hours were measured with a gas chromatograph, the oxygen concentration was 0.55% by volume and the carbon dioxide concentration was 10.7% by volume. Further, when the oxygen concentration and the carbon dioxide concentration after 24 hours were measured with a gas chromatograph, the oxygen concentration was 0.00% by volume and the carbon dioxide concentration was 11.0% by volume.

Claims (5)

(e) at least one selected from the group consisting of alkali metal carbonates, alkali metal hydroxides and alkaline earth metal hydroxides, (a) dehydroascorbic acid, (c) a transition metal catalyst, (d) activated carbon , and (f) water,
(b) does not contain ascorbic acids, or (b) further contains ascorbic acids, and the molar ratio [(b)/(a)] of (b) ascorbic acids and (a) dehydroascorbic acid is 1.5 or less A gas concentration regulator for anaerobic bacteria culture. (b) The gas concentration adjusting agent for culturing anaerobic bacteria according to claim 1, containing ascorbic acids. 3. The gas concentration adjusting agent for culturing anaerobic bacteria according to claim 2, wherein the molar ratio [(b)/(a)] of (b) ascorbic acids and (a) dehydroascorbic acid is 1.2 or less. A method for culturing anaerobic bacteria, comprising culturing anaerobic bacteria in the presence of the gas concentration adjusting agent according to any one of claims 1 to 3. A gas concentration adjusting agent package in which the gas concentration adjusting agent according to any one of claims 1 to 3 is packaged in a small bag with an air-permeable packaging material.